Preparation of cell-permeable Cre recombinase by expressed protein ligation

Background

Protein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli.

Results

Expressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC.

Conclusions

Collectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0126-z) contains supplementary material, which is available to authorized users.     

Coordination conjugates of therapeutic proteins with drug carriers: a new approach for versatile advanced drug delivery

We present here a general system for the coordination attachment of therapeutic proteins to a drug delivery system and its application in combined therapy. Proof of concept is demonstrated by the synthesis and testing of the targeted drug delivery system for cytostatics, which is based on a combination of the drug carrier Zn-porphyrin-cyclodextrin conjugates and their supramolecular coordination complexes with immunoglobulins. This system can be as readily used for a variety of therapeutic and targeting proteins including PAs, MAs, lectins, and HSA. Moreover, it allows combined photodynamic therapy, cell targeted chemotherapy and immunotherapy. When tested in a mouse model with human C32 carcinoma, the therapeutic superiority of the coordination assembly nanosystem was shown in comparison with the efficacy of building blocks used for the construction of the system.     

普贝生用于足月妊娠促宫颈成熟的疗效观察

目的：探讨普贝生促进宫颈成熟,提高足月妊娠经阴道分娩的有效性和安全性。方法：120例足月妊娠的未临产孕妇随机分为试验组与对照组,其中试验组50例给予阴道后穹隆置入普贝生1～2次,对照组50例给予小剂量催产素静脉滴注,比较两组宫颈成熟度,分娩情况及对于产妇、新生儿的影响。结果：①试验组宫颈Bishop评分增加3．81±1．04,对照组增加3．09±1．15,两组间差异有统计学意义（P〈0．05）②试验组促宫颈成熟的显效率为78．33％,总有效率为91．67％,高于对照组35．00％显效率和63．33％总有效率（P〈0．01）。③试验组阴道分娩率73．33％,进入产程时间（34．19±13．20）h,产程（8．47±2．68）h,对照组阴道分娩率41．67％,进入产程时间（52．14±16．05）h,产程（12．25±3．73）h,两组间比较差异有显著性（P〈0．01或0．05）。④试验组产后出血量（225．31±67．80）ml,新生儿体重（3369．48±311．65）g,Apgar评分9．52±0．39,对照组产后出血量（232．44±75．76）ml,新生儿体重（3417．63±359．68）g,Apgar评分9．48±0．47,两组间差异无统计学意义（P〉0．05）。结论：普贝生可有效促进足月妊娠产妇的宫颈成熟．提高经阴道引产成功率,降低剖宫产率,且安全性好,对母儿影响小。     

Microfabricated particulate drug-delivery systems

Micro- and nanoparticulate drug-delivery systems (DDSs) play a significant role in formulation sciences. Most particulate DDSs are scaffold-free, although some particles are encapsulated inside other biomaterials for controlled release. Despite rapid progress in recent years, challenges still remain in controlling the homogenicity of micro-/nanoparticles, especially for two crucial factors in particulate DDSs: the size and shape of the particles. Recent approaches make use of microfabrication techniques to generate micro-/nanoparticles with highly controllable architectures free of scaffolds. This review presents an overview of a burgeoning field of DDSs, which can potentially overcome some drawbacks of conventional techniques for particle fabrication and offer better control of particulate DDSs.